The term "lentiviral vectors" or "SIVagm vectors" refers to infectious, but propagation-incompetent retroviruses capable of introducing genes into cells in the form of retroviral expression vectors (also called expression constructs or packaging-competent constructs). Lentiviruses refers to a group of retroviridae which following an infection of man, other primates, and mammals (e.g. sheep, cats) leads to a disease condition after a long incubation period. Gene transfer using retroviruses or lentiviruses, respectively, is also referred to as transduction. The gene transfer results in an integration of the expression vector into the cellular genome. Expression vectors include a packaging signal psi leading to incorporation of RNA of the expression vector into vector particles and to gene transfer. Therefore, "psi" refers to the retroviral packaging signal controlling efficient packaging of messenger RNA of the expression vector. Furthermore, the expression vector must be flanked by lentiviral LTR sequences ("long terminal repeats") in order to enable correct transcription of the RNA of the expression vector into DNA and subsequent integration of the expression vector gene into the chromosomal DNA of the cell. Lentiviral gene transfer is advantageous because (i) generally a copy of the desired gene is transduced into cells, (ii) usually, the gene is transferred without mutations or rearrangements, (iii) stable incorporation into the chromosome occurs, and (iv) genes can also be transferred into non-proliferating cells.
It has been known to use lentiviral vectors on the basis of the human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), and the simian immunodeficiency virus of the rhesus monkey (Macaca mulatta) for the transfer of particular genes into mammalian cells and also specifically into human cells. A particular advantage of lentiviral vectors is their ability to transduce also resting or non-dividing cells, respectively. These vectors are propagation-incompetent and merely go through one cycle of replication. Three components are required for the preparation of such vectors. Within a packaging cell, a psi-negative gag/pol gene of the original lentivirus, a psi-negative env gene which may be derived from lentiviruses or other viruses, and a psi-positive and therefore packaging-competent expression construct usually also derived from a lentivirus. The expression vector enables packaging into the retroviral vector and transfer by the retrovirus to transduce a coding and translation-competent region of the desired gene product into the target cell. Following transduction of the plasmids containing the gag/pol gene, the env gene, and the expression vector gene by transfection of the three respective DNAs into a suitable mammalian cell, a packaging cell is generated which releases retroviral vector particles into the cell supernatant which exclusively contain the expression construct, however, lacking the psi-negative gag/pol and env genes so that these are not introduced into the target cells.
The tropism of lentiviral vectors, i.e. the selection of the mammalian cells into which they are able to transduce the expression construct is determined by the env gene in the packaging cell used and, thus, by the env gene products present in the vector particles. The env gene of retroviruses among which e.g. the murine leukemia virus (MLV), several lentiviruses such as HIV, SIV, or FIV ("feline immunodeficiency virus"), but also EIAV ("equine infectious anemia virus") or CIAV ("caprine infectious anemia virus") are used in the formation of lentiviral vector particles is translated into envelope proteins, the transmembrane protein (TM) and the surface envelope protein (SU) forming the outer envelope of the lentiviral vector. To date, mainly the env gene products of the amphotropic MLV, the GaLV ("gibbon ape leukemia virus") and the G protein of VSV ("vesicular stomatitis virus"; Burns et al., Proc. Natl. Acad. Sci. USA 90 (1993), 8033-8037) are used for gene transfer. They enable gene transfer into a wide variety of different mammalian cells and also in human cells. Particularly for selective gene transfer into human cells of a specific cell type, e.g. T cells or hematopoietic stem cells, the env gene products of the ecotropic, the amphotropic MLV or the spleen necrosis virus (SNV) are useful if they have been modified by introduction of domains of single chain antibodies (scFv, "single chain Fv") or other ligands for cell surface proteins such as for example cytokins or growth factors.
To improve the transduction of various genes, retroviral vectors have been proposed in which the gag/pol gene of different lentiviruses such as HIV-1, HIV-2, SIVmac, FIV, or EIAV was used instead of the gag/pol gene of oncoretroviruses such as MLV. Therefore, it is an object of the present invention to provide improved lentiviral vectors (retroviral virus particles).
This object has been achieved by the claimed invention.